DEVELOPMENT OF POROUS DEFECTS IN PLASMA-MEMBRANES OF ADENOSINE TRIPHOSPHATE-DEPLETED MADIN-DARBY CANINE KIDNEY-CELLS AND ITS INHIBITION BY GLYCINE

Studies during the past decade have led to the recognition of a fundam ental, widely expressed mechanism of structural damage in energy-depri ved cells, which is suppressed by physiologic levels of glycine and is independent of Ca2+-availability or alterations of cytosolic free Ca2 +. To gain insight into this process, Madin-Darby canine kidney (MDCK) cells were depleted of adenosine triphosphate (ATP) by a mitochondria l uncoupler in glucose-free medium, and intracellular free Ca2+ was cl amped at 100 nM to avoid calcium cytotoxicity. Although the ATP-deplet ed cells swelled and blebbed and their plasma membranes appeared to be under tension, they nevertheless became permeable to macromolecules. The plasma membranes of these cells retained structural continuity, as determined by morphologic observations, and confocal microscopy of a plasma membrane protein label (Biotin: Ultra Avidin-Texas Red) and a l ipid label (NBD-sphingomyelin). Using fluoresceinated dextrans of grad ed molecular size, membrane permselectivity was examined noninvasively by confocal microscopy. Measured as inside/outside ratios of fluoresc ence intensity, the permeability indices showed progressively greater restriction to diffusion of increasingly larger dextran molecules acro ss plasma membranes, with sharp break-points between 70,000 and 145,00 0 daltons (d). The results indicated that the membranes behaved as if they were perforated by water-filled channels or ''pores,'' with size- exclusion limits of molecular dimensions. The membrane defects evolved from small pores permeable only to propidium iodide (668 d) and the s mallest dextran (4,000 d), before enlarging with time to become permea ble to larger dextrans. Inclusion of glycine during ATP depletion did not affect cell swelling or blebbing but completely prevented the deve lopment of permeability defects. Treatment of cells before ATP depleti on with a membrane-impermeant homobifunctional ''nearest neighbor'' cr osslinking agent, 3,3' dithiobis(sulfosuccinimidylpropionate), suppres sed the development of permeability defects, even in the absence of gl ycine. These observations suggest that the cellular abnormality that i s suppressed by glycine involves rearrangement of plasma membrane prot eins to form water-filled pores large enough to leak macromolecules.